In vitro study of the NS2-3 protease of hepatitis C virus.

Processing at the C terminus of the NS2 protein of hepatitis C virus (HCV) is mediated by a virus-encoded protease which spans most of the NS2 protein and part of the NS3 polypeptide. In vitro cotranslational cleavage at the 2-3 junction is stimulated by the presence of microsomal membranes and ultimately results in the membrane insertion of the NS2 polypeptide. To characterize the biochemical properties of this viral protease, we have established an in vitro assay whereby the NS2-3 protease of HCV BK can be activated posttranslationally by the addition of detergents. The cleavage proficiency of several deletion and single point mutants was the same as that observed with microsomal membranes, indicating that the overall sequence requirements for proper cleavage at this site are maintained even under these artificial conditions. The processing efficiency of the NS2-3 protease varied according to the type of detergent used and its concentration. Also, the incubation temperature affected the cleavage at the 2-3 junction. The autoproteolytic activity of the NS2-3 protease could be inhibited by alkylating agents such as iodoacetamide and N-ethylmaleimide. Metal chelators such as EDTA and phenanthroline also inhibited the viral enzyme. The EDTA inhibition of NS2-3 cleavage could be reversed, at least in part, by the addition of ZnCl2 and CdCl2. Among the common protease inhibitors tested, tosyl phenylalanyl chloromethyl ketone and soybean trypsin inhibitor inactivated the NS2-3 protease. By means of gel filtration analysis, it was observed that the redox state of the reaction mixture greatly influenced the processing efficiency at the 2-3 site and that factors present in the rabbit reticulocyte lysate, wheat germ extract, and HeLa cell extract were required for efficient processing at this site. Thus, the in vitro assay should allow further characterization of the biochemical properties of the NS2-3 protease of HCV and the identification of host components that contribute to the efficient processing at the 2-3 junction.     

Neurochemical effects ofl-pyroglutamic acid

The effect ofl-pyroglutamic acid, a metabolite that accumulates in pyroglutamic aciduria, on different neurochemical parameters was investigated in adult male Wistar rats. Glutamate binding, adenylate cyclase activity and G protein coupling to adenylate cyclase were assayed in the presence of the acid.l-pyroglutamic acid decreased Na⁺-dependent and Na⁺-independent glutamate binding Basal and GMP-PNP stimulated adenylate cyclase activity were not affected by the acid. Furthermore, rats received unilateral intrastriatal injections of 10–300 nmol of bufferedl-pyroglutamic acid. Vehicle (0.25 M Tris-Cl, pH 7.35–7.4) was injected into the contralateral striatum. Neurotoxic damage was assessed seven days after the injection by histological examination and by weighing both cerebral hemispheres. No difference in histology or weight could be identified between hemispheres. These results suggest that, although capable of interfering with glutamate binding, pyroglutamate did not cause a major lesion in the present model of neurotoxicity.     

Vasoactive factors in decompensated cirrhosis. Effect of acute plasma expansion

Plasma volume expansion was performed in 16 cirrhotic patients with ascites, 8 with avid sodium retention (sodium retainers) and 8 with normal sodium balance (sodium excretors). No natriuretic response was observed in sodium retainers (daily UNa = 7.1 +/- 1.5 mEq before expansion and 20.8 +/- 7.8 after expansion; p = not significant). After expansion plasma renin activity and plasma aldosterone showed a fall in both groups, whereas urinary kallikrein excretion decreased significantly in sodium retainers (27.1 +/- 9.7 before expansion and 7.8 +/- 6.4 after expansion; p less than 0.05). Baseline PGE were higher than normal in sodium retainers (997.0 +/- 134.3; p less than 0.02 vs. controls) and increased after expansion. Plasma octopamine was always within normal range. These results suggest that: a) reduction of effective plasma volume is not the main factor involved in sodium retention; b) the renin-angiotensin-aldosterone system has only a permissive role; c) prostaglandin system is activated and could have a protective role in maintaining renal function in cirrhotic patients.     

Administration of semisynthetic human insulin by a spray injector

The use of Jet injection in insulin administration pointed out the question whether this route could affect insulin absorption and plasma insulin profiles. To compare plasma insulin profiles following an administration of an identical insulin dose by jet injection or by conventional subcutaneous route (syringe with needle) 8 healthy subjects (age 24-28 yrs., non obese) were given at 09.00 h of two different days 200 mU/kg/BW of human semisynthetic regular insulin (Novo Actarapid) alternatively subcutaneously by a syringe with needle or transcutaneously by jet injection (DG 77 - Sicim - Gorizia). Before insulin administration and then 15, 30, 60, 90, 120 and 180 minutes after, blood samples were drawn for plasma insulin and C-peptide determination. Higher plasma insulin levels after administration by jet were found at 15' and 30' minutes (62,58 +/- 6,31 v.s. 36,94 +/- 3,31 microunits/ml at 15' and 76,51 +/- 9,60 v.s. 51,65 +/- 9,95 at 30', p less than 0,01 and p less than 0,005, paired Student t test). No difference could be observed for the other times. C-peptide was found to fall to undetectable values, confirming the nearly total suppression of endogenous insulin production. It is concluded that total regular insulin absorption does not differ after transcutaneous jet injection or administration by syringe with needle, but in the first case it is faster. This last finding should be considered in planning insulin treatment schedules.     

Action of veratridine on acetylcholinesterase in cultures of rat muscle cells

Studies on cultures of embryonic rat muscle cells have suggested that the presence of collagen-tailed forms may be correlated with spontaneous contractile activity: these forms disappear in the presence of tetrodotoxin which blocks the sodium channels involved in the propagation of action potentials. The effect of veratridine, a drug which maintains the sodium channels in the open state, was studied. It is shown here that in young cultures veratridine provoked a dramatic increase in total acetylcholinesterase activity and changed the distribution of the molecular forms of the enzyme, increasing the proportion and absolute amount of the A12 form. In order to elucidate the mechanism of action of this drug, the effects of various ions, ionophores, or other agents that modify the ionic permeabilities of membranes were also investigated.     

Nitrogen fixation efficiency,natural15N abundance,and morphology of mesquite (Prosopis glandulosa) root nodules

Summary The relative nitrogen fixation efficiencies (RE 1-[H₂ evolved÷C₂H₂ reduced]·100) of four mesquite (Prosopis glandulosa var.torreyana) rhizobia (Strains WR 1001, WR 1002, L5, L9) and a cowpea rhizobia (Strain 176A32) on mesquite were evaluated in a glasshouse experiment. Plant yield, shoot N accumulation, and the natural¹⁵N abundance (¹⁵N) of nodule tissue were determined. Strain WR 1002 failed to nodulate mesquite and strain L5 produced ineffective nodules. Among the three effective strains (WR 1001, L9, 176A32) the cowpea strain (176A32) and strain L9 had significantly higher RE than strain WR 1001. Differences in RE, however, were not accompanied by significantly higher plant yield and shoot N accumulation. The difference in¹⁵N abundance between foliar tissue and nodules (nodules minus leaves) was 0.47 ¹⁵N for the ineffective L5 nodules, while for the effective WR 1001, L9, and 176A32 nodules, respectively, this difference was 8.35, 7.81, and 8.35 ¹⁵N. This indicates a similar relationship between N₂-fixing effectiveness and natural¹⁵N enrichment of nodules that was previously observed in soybeans (Glycine max, L. Merr.). Strains WR 1001 and L9 produced elongate, indeterminate nodules typical for mesquite. The ineffective L5 nodules had few infected cells and an abundance of cortical amyloplasts. Mesquite nodules produced by the cowpea strain were spherical and were somewhat more similar in internal morphology to determinate nodules typical of cowpea than indeterminate nodules normally associated with mesquite.     

Assembly of Bacillus subtilis phage phi29. 1. Mutants in the cistrons coding for the structural proteins.

The effect of mutations in the cistrons coding for the phage structural proteins has been studied by analyzing the phage-related structures accumulated after restrictive infection. Infection with susmutants in cistron 8, lacking both the major head and the fiber protein, does not produce any phage-related structure, suggesting a single route for the assembly of phage phi29; infection with ts mutants in this cistron produces isometric particles. Mutants is cistron 9, coding for the tail protein, TP1, produce DNA-free prolate heads with an internal core; these particles are abortive and contain the head proteins HPO, HP1 and HP3, the upper collar protein NP2 and the nonstructural proteins p7, p15 and p16. Mutants in cistron 10, coding for the upper collar protein, NP2, produce DNA-free isometric heads also with an internal core; they contain the head proteins and the nonstructural protein p7, suggesting that this protein forms the internal core. Mutants in cistrons 11 and 12, coding for the lower collar protein, NP3, and the neck appendages, NP1, respectively, give rise to the formation of DNA-containing normal capsids and DNA-free prolate particles, more rounded at the corners than the normal capsids and with an internal core; the DNA-containing 11-particles are formed by the head proteins and the upper collar protein; the DNA-free 11-particles contain, besides these proteins, the nonstructural protein p7 and a small amount of proteins p15 and 16. The DNA-containing 12-particles have all the normal phage structural proteins except the neck appendages, formed by protein NP1; the DNA-free particles are similar to the DNA-free 11-particles. After restricitive infection mutant sus14(1241) has a delayed lysis phenotype and produces a phage burst higher than normal, after artificial lysis. It produces DNA-containing particles, identical to wild-type phage, which have all the normal phage structural proteins, and DNA-free prolate particles, more rounded at the corners than the final phage particles and with an internal core; the last particles contain the same proteins as the DNA-free 11 or 12-particles. These particles could represent a prohead state, ready for DNA encapsulation. None of the DNA-containing particles have the nonstructural proteins p7, p15 or p16, suggesting that these proteins are released from the proheads upon DNA encapsulation.     

2-fluorourocanic acid, a potent reversible inhibitor of urocanase.

The K_i for the interaction of 2-fluorourocanic acid with urocanase (from $\text{Pseudomonas fluorescens}$) is 1000 times as great as K_m for the natural substrate, urocanic acid, whereas enzymatic hydration of the fluoro analog occurs $\text{ca}$. 100 times more slowly. Inhibition is competive and is eventually overcome by utilization of the analog. By contrast, 4-fluoro- and 2-amino-urocanic acid are neither significant inhibitors nor substrates for the enzyme. 2-Fluorourocanic acid may prove a useful tool for blocking the utilization of histidine as a one-carbon source in metabolism.     

A research on the action of testosterone propionate and of ciproterone acetate on the mouse-killing behaviour of the adult rat.

Data demonstrate exactly that testosterone favour above all the killing behaviour of castrate males at birth in comparison with control males while ciproterone acetate turns out to be more effective in diminishing the killing behaviour of males having integral testicle and of androgenized females at birth in comparison with castrated males. The results point out that the killing behaviour of rats is affected by the presence of testosterone in the first days of life, and in the grown-up animal.     

Isolated Aortocoronary Bypass Operations in Patients Over 70 Years of Age: A Comparison With Results in Patients 50 to 59 Years Old

The early and late morbidity, mortality and beneficial effects of isolated aortocoronary bypass operations in a group of 35 patients 70 years old or older were compared with those factors in patients 50 to 59 years old. The patients in both groups were matched according to the year in which the operation was done and the number of vessels bypassed. Left ventricular function, estimated by the angiographically calculated ejection fraction, was not statistically different in the two groups. Cardiac index, while adequate in both groups, was significantly lower in the older age group. Comparisons were made of “early” events, such as perioperative myocardial infarction, perioperative death and length of post-operative hospital stay; and of “late” events, including myocardial infarction, angina pectoris, congestive heart failure and death, which occurred after patients were discharged from the hospital. The mean length of follow-up of patients was similar in both groups.In comparing early events in the two groups, there was no statistically significant difference in the incidence of perioperative myocardial infarction, perioperative mortality or mean length of postoperative hospital stays. With regard to late events, there was no statistically significant difference in the incidences of myocardial infarction, angina pectoris or mortality.